Proteomic profiling identifies breast tumor metastasis‐associated factors in an isogenic model

A combination of LC and MS was applied to an isogenic breast tumor metastasis model to identify proteins associated with a cellular phenotype. Chromatofocusing followed by nonporous‐RP‐HPLC/ESI‐TOF MS was applied to cell lysates of a pair of monoclonal cell lines from the human breast carcinoma cell line MDA‐MB‐435 that have different metastatic phenotypes in immune‐compromised mice. This method was developed to separate proteins based on pI and hydrophobicity. The high resolution and mass accuracy of ESI‐TOF measurements provided a good correlation of theoretical MW and experimental Mr values of intact proteins measured in mass maps obtained in the pH range 3.8–6.4. The isolated proteins were digested by trypsin and analyzed by MALDI‐TOF MS, MALDI‐QIT‐TOF MS, and monolith‐based HPLC/MS/MS. The unique combination of the techniques provided valuable information including quantitation and modification of proteins. We identified 89 selected proteins, of which 43 were confirmed as differentially expressed. Metastasis‐associated proteins included galectin‐1, whereas annexin I and annexin II were associated with the nonmetastatic phenotype. In this study, we demonstrate that combining a variety of MS tools with a multidimensional liquid‐phase separation provides the ability to map cellular protein content, to search for modified proteins, and to correlate protein expression with cellular phenotype.